Methods previously known for transformation of monocotyledons such as maize and rice, which are major grain crops, include electroporation, particle gun transformation, etc. However, these physical gene transfer methods have problems in that genes are introduced as multiple copies or are not inserted in an intact state, and the resulting transformed plants may often develop malformations and sterility.
Agrobacterium-mediated gene transfer is universally used as a transformation method for dicotyledons. Although it has been understood that hosts of Agrobacterium are limited only to dicotyledons and Agrobacterium has no ability to infect monocotyledons (De Cleene and De Ley 1976), some attempts have been made to transform monocotyledons through Agrobacterium-mediated method.
Grimsley et al. have reported that when maize streak virus DNA was inserted into T-DNA of Agrobacterium and inoculated into maize growing points, infection with maize streak virus was confirmed. Since such infection symptoms are not observed simply when the maize streak virus DNA alone is inoculated, Grimsley et al. have recognized that the above observation indicates the ability of Agrobacterium to introduce DNA into maize (Grimsley et al. 1987). However, this result is not indicative of T-DNA integration into nuclei, because a virus will multiply even when not integrated into a nuclear genome. Grimsley et al. have further demonstrated that the highest infection efficiency is observed upon inoculation into a growing point in the shoot apex of maize (Grimsley et al. 1988), and that the VirC gene in plasmids of Agrobacterium is essential for infection (Grimsley et al. 1989).
Gould et al. injured maize growing points with a needle and then inoculated these growing points with super-virulent Agrobacterium EHA1 carrying the kanamycin resistance gene and the GUS gene, followed by kanamycin selection on the treated growing points to obtain a resistant plant. Upon Southern analysis to confirm whether progeny seeds of this plant have the introduced gene, they confirmed that some seeds had the transgene (Gould et al. 1991). This indicates that the whole plant obtained by kanamycin selection on Agrobacterium-treated growing points had both transformed and non-transformed cells (chimerism).
Mooney et al. attempted to introduce the kanamycin resistance gene into wheat embryos by using Agrobacterium. First, the embryos were enzymatically treated to injure their cell walls, and then inoculated with Agrobacterium. Among the treated calli, very few calli were grown that appeared to be resistant to kanamycin, but no whole plant was regenerated from these calli. Upon Southern analysis to confirm the presence of the kanamycin resistance gene, all the resistant calli were found to have a structural mutation in the transgene (Mooney et al. 1991).
Raineri et al. performed super-virulent Agrobacterium A281 (pTiBo542) treatment on 8 varieties of rice whose scutellum had been injured, and they confirmed tumorous tissue growth in 2 varieties of Nipponbare, Fujisaka 5. Further, when rice embryos were inoculated with Agrobacterium carrying a Ti plasmid modified to have the kanamycin resistance gene and the GUS gene wherein hormone synthesis genes in T-DNA have been removed, the growth of kanamycin-resistant calli was observed. In these resistant calli, GUS gene expression was observed, but no transformed plant was obtained. Based on these results, Raineri et al. have recognized that the Agrobacterium T-DNA was introduced into rice cells (Raineri et al. 1990).
As shown above, there are study reports suggesting that Agrobacterium-mediated gene transfer is also possible for Gramineae crops including rice, maize and wheat, but these reports failed to show persuasive results because these studies had a problem in reproducibility and were also insufficient for transgene confirmation (Potrykus 1990).
Chan et al. injured immature rice embryos, which had been cultured for 2 days in the presence of 2,4-D, and then inoculated these embryos with Agrobacterium carrying genes for npt II and GUS in a medium containing suspension-cultured potato cells. They cultured the thus treated immature embryos on a G418-containing medium to obtain regenerated plants from the induced calli. They confirmed the location of the GUS gene in the regenerated plants and their progeny plants by Southern analysis, and reported that the presence of the transgene was observed in plants of both R0 and R1 generations (Chan et al. 1993). This result supports Agrobacterium-mediated transformation in rice, but the transformation efficiency was as low as 1.6%. Moreover, there was only one regenerated plant that showed normal growth, although 250 immature embryos were used for testing. Since enormous efforts are required to extract immature embryos of rice, such low transformation efficiency is not practical.
In recent years, it has been reported that stable and highly efficient transformation is also possible in monocotyledons including rice and maize when using a super-binary vector carrying a part of the virulence gene from super-virulent Agrobacterium (Hiei et al. 1994, Ishida et al., 1996). These reports suggest that Agrobacterium-mediated transformation not only allows stable and highly efficient transformation, but is also advantageous in that the resulting transformed plants have fewer mutations, and in that the introduced genes are low in copy number and are often in an intact state. Following success in rice and maize, further reports were issued for Agrobacterium-mediated transformation in other major grain crops, i.e., wheat (Cheng et al. 1997), barley (Tingay et al. 1997) and sorghum (Zhao et al. 2000).
In addition to the above, other attempts have been made to improve the efficiency of Agrobacterium-mediated maize transformation, as exemplified by selection of transformed cells on N6 basal medium (Zhao et al. 2001); addition of AgNO3 and carbenicillin to culture medium (Zhao et al. 2001, Ishida et al. 2003); and addition of cysteine to coculture medium (Frame et al. 2002). As shown above, modifications to the medium composition or selection marker genes also result in improved efficiency of Agrobacterium-mediated transformation in rice and maize.
However, in almost all the methods previously reported for Agrobacterium-mediated transformation in rice or maize, a transformed plant is obtained from a callus derived from an Agrobacterium-inoculated scutellum or immature embryo through the steps of allowing a transformed callus to selectively grow on a medium containing a herbicide component or an antibiotic, and placing the resulting transformed cell clump onto a regeneration medium to induce regeneration (Deji et al., 2000; Negrotto et al., 2000; Nomura et al., 2000a; Nomura et al., 2000b; Taniguchi et al., 2000; Frame et al., 2002; Zhang et al., 2003; Frame et al. 2006).
In plant transformation methods, selection of transformed cells is imperative for production of transformed plants, and it has been believed that plant transformation cannot succeed in the absence of this step (Potrykus et al., 1998; Erikson et al., 2005; Joersbo et al., 2001). In most cases, selection of transformed cells is accomplished as follows: a plant material is introduced with a gene resistant to a drug that inhibits proliferation of non-transformed cells, and then cultured with a medium containing this drug, whereby only transformed cells expressing the drug resistance gene integrated into the plant cell genome are allowed to selectively proliferate.
Among genes used for selection of transformed cells (selection marker genes), the most commonly used are genes conferring resistance to herbicides or antibiotics (Kuiper et al. 2001). Genes conferring resistance to herbicides include the bar gene and the EPSP gene (De Block et al., 1987; Comai et al., 1985), while genes conferring resistance to antibiotics include the NPTII gene and the HPT gene (Bevan et al., 1983; Waldron et al., 1985), each of which is often used as a selection marker gene for plant transformation. Moreover, recent reports have shown that genes conferring the ability to metabolize a specific sugar(s), e.g., the PMI gene and the Xy1A gene (Joersbo et al., 1998; Haldrup et al., 1998) are also effective as selection marker genes. As selection marker genes based on the mechanism for allowing transformed cells to selectively proliferate, many genes have been reported in addition to those mentioned above. Moreover, in transformation methods using these genes, a selection step of allowing transformed cells to selectively proliferate is regarded as essential.
In the case of in planta transformation in which flower bud tissue is transformed by immersion under reduced pressure, transformed seeds are obtained without any selection step (Bent, 2000). However, to obtain desired transformed seeds from a mixture containing many non-transformed seeds, a selection step using an antibiotic resistance gene or the like is required.
There are reports of a method which involves introducing the GFP gene and visually screening transformed sites by detection of cells exhibiting fluorescence under ultraviolet irradiation to obtain a transformed plant (Elliott et al., 1998; Zhu et al., 2004). This method does not select transformed cells from a mixture containing non-transformed cells based on differences in their growth, but it requires a selection step for distinguishing and isolating transformed cells from non-transformed cells based on the presence or absence of GFP gene expression.
Techniques reported for removing a selection marker gene from a transformed plant include a co-transformation system (Komari et al., 1996), a MAT vector system (Ebinuma et al., 1997) and a CreLox system (Gleave et al., 1999; Zhang et al., 2003). When using these systems, it is possible to obtain a transformed plant free from a selection marker gene. However, in the course of the production of a selection marker-free transformed plant, it is necessary to perform a step of distinguishing and isolating transformed cells from non-transformed cells by using a conventionally used drug resistance gene or plant hormone synthesis gene, etc.
As shown above, the methods previously used for plant transformation must involve a selection step for isolating transformed cells from non-transformed cells. This selection step requires a selection marker gene used for selection purposes, in addition to a gene of interest (GOI gene), as shown above. By means of a reaction caused by the action of a protein or enzyme produced upon expression of this selection marker gene (e.g., herbicide resistance, antibiotic resistance or fluorescence emission), a very few transformed cells among many non-transformed cells can be distinguished and grown to obtain a transformed plant. However, such a selection marker gene is no longer required for a produced transformed plant, and many common consumers are insecure about the use of transformed plants because when selection marker genes remain in the transformed plants, the risk of spreading herbicide resistance genes or antibiotic resistance genes to normal non-recombinant plants via transformants is not negligible. Moreover, many types of selection marker genes are reported, but they have limited use depending on the species of plants, which will cause a problem when multiple genes are introduced separately. Further, although some techniques are reported for removing a selection marker gene from a transformant, these techniques require enormous efforts, including use of a longer culture period than that of standard transformation and/or isolation of selection marker-free plants among progeny plants.
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